Circadian regulator BMAL1::CLOCK promotes cell proliferation in hepatocellular carcinoma by controlling apoptosis and cell cycle

Significance The circadian clock modulates the expression of many protein-coding genes in most cell types, thereby playing a key role in human health. Recent discoveries have unveiled the circadian clock as a novel pathway for therapeutic intervention in cancer. Liver cancer remains a major public health concern globally and is closely associated with dysregulated circadian parameters. However, the underlying mechanism is currently unknown. Our findings establish anti-apoptotic roles of the master clock regulators BMAL1 and CLOCK in promoting proliferation of liver cancer cells and reveal an underpinning mechanism being mediated by the cancer-state essential gene Wee1.

Hepatocellular carcinoma (HCC) remains a global health challenge whose incidence is growing worldwide. Previous evidence strongly supported the notion that the circadian clock controls physiological homeostasis of the liver and plays a key role in hepatocarcinogenesis. Despite the progress, cellular and molecular mechanisms underpinning this HCC-clock crosstalk remain unknown. Addressing this knowledge gap, we show here that although the human HCC cells Hep3B, HepG2, and Huh7 displayed variations in circadian rhythm profiles, all cells relied on the master circadian clock transcription factors, BMAL1 and CLOCK, for sustained cell growth. Down-regulating Bmal1 or Clock in the HCC cells induced apoptosis and arrested cell cycle at the G 2 /M phase. Mechanistically, we found that inhibiting Bmal1/Clock induced dysregulation of the cell cycle regulators Wee1 and p21 which cooperatively contribute to tumor cell death. Bmal1/Clock knockdown caused downregulation of Wee1 that led to apoptosis activation and upregulation of p21 which arrested the cell cycle at the G 2 /M phase. Collectively, our results suggest that the circadian clock regulators BMAL1 and CLOCK promote HCC cell proliferation by controlling Wee1 and p21 levels, thereby preventing apoptosis and cell cycle arrest. Our findings shed light on cellular impact of the clock proteins for maintaining HCC oncogenesis and provide proof-of-principle for developing cancer therapy based on modulation of the circadian clock.
circadian clock | hepatocellular carcinoma | cell cycle | apoptosis Hepatocellular carcinoma (HCC), the most common form of liver cancer, is the third leading cause of cancer-related deaths worldwide. As the incidence of viral hepatitis-related liver cancer has declined due to preventive interventions and treatments, other etiologies such as non-alcoholic fatty liver disease (NAFLD) and alcohol-associated liver disease (ALD) are emerging as leading risk factors associated with rising HCC cases (1). Molecularly targeted therapeutics have demonstrated efficacy for HCC treatment; the combination of atezolizumab (anti-PD-L1 antibody) and bevacizumab (anti-vascular endothelial growth factor antibody) has become the new standard first-line treatment demonstrating superior survival and response rate compared to the prior standard of sorafenib (2). While these agents have the potential to improve patient outcomes, the efficacy is far from adequate, and the objective response rates are still low (about 25%) (3). Defining the underlying molecular mechanisms driving HCC initiation and growth is urgently needed to lay the foundation for novel molecularly targeted therapeutic approaches.
The circadian clock represents an evolutionarily conserved mechanism that achieves the coordination of physiological processes and behavior with the day-night cycles. The molecular clock centered on the transcription factor BMAL1 and its partner CLOCK (BMAL1::CLOCK) establishes rhythmic expression of most protein-coding genes in mammalian genomes, thereby gating key physiological outputs, including sleep, feeding, hormone secretion, metabolism, and immune responses (4,5). Several studies have demonstrated that core clock genes, including Bmal1 and Clock, are commonly dysregulated or mutated in cancer cells (6,7). Epidemiological and genetic studies have uncovered reciprocal regulation of disrupted circadian homeostasis and oncogenic processes in the context of several cancer types (8)(9)(10)(11)(12)(13)(14). Importantly, modulation of the core clock by small molecules has recently emerged as a promising new approach in cancer therapy, thus providing a possible path toward experimental therapeutics (10,14).
Due to the distinct chromatin landscape shaped by tissue-specific pioneer factors, the liver represents a physiological hub for circadian regulation (4,15,16). The hepatic circadian transcripts are involved in principal functions of the liver, including glucose homeostasis, lipogenesis, bile acid synthesis, mitochondrial biogenesis, oxidative metabolism, amino acid turnover, and xenobiotic detoxification (17). Chronic jet lag disturbing the circadian rhythms accelerated diethylnitrosamine (DEN)-induced liver carcinogenesis (18)   caused spontaneous HCC development following increased susceptibility to NAFLD (19). Alterations of the circadian clock genes are correlated with survival and clinical outcome of HCC patients (6). Accordingly, the circadian control of liver physiological homeostasis plays a key role in hepatocarcinogenesis. Despite the genetic correlations, the molecular functions of the clock genes implicated in HCC pathogenesis are still enigmatic, which hampers the efforts to leverage therapeutic strategies targeting clock proteins in HCC.
In the present study, we report that the core clock genes Bmal1 and Clock play a pro-proliferative role in HCC by controlling cell cycle regulators. Down-regulating Bmal1 and Clock induced HCC cytotoxicity, activated apoptosis, and altered cell cycle progression. Wee1 encoding an essential kinase in the G 2 /M checkpoint and p21 Cdkn1a/Waf1/Cip1 (referred to as p21 hereafter) encoding a cyclin-dependent kinase inhibitor that negatively regulate cell cycle progression are transcriptional targets of the circadian clock circuit that contribute to Bmal1/Clock-promoted HCC cell proliferation. Bmal1/Clock knockdown induced downregulation of Wee1 leading to enhanced apoptosis. However, the G 2 /M phase arrest caused by Bmal1/Clock knockdown was independent of Wee1 but associated with altered expression of the p21 gene. Therefore, Bmal1 and Clock control HCC cell proliferation through divergent molecular mechanisms that involve the inhibition of apoptosis and cell cycle arrest.

Results
The Core Clock Genes Bmal1 and Clock Are Indispensable for HCC Cell Growth. We first interrogated the circadian rhythms in three widely used human HCC cells, Hep3B, HepG2, and Huh7, finding each displayed a distinct pattern ( Fig. 1 A-C), with the Hep3B cells oscillating most robustly and the Huh7 cells not technically cycling according to circadian parameters (SI Appendix, Fig. S1). To study the functional roles of the core circadian clock in the HCC cells, we targeted Bmal1 and Clock by introducing siRNA-mediated gene knockdown. Despite the heterogeneous intrinsic circadian oscillations, targeting either Bmal1 or Clock potently impaired the proliferation of all three HCC cell lines ( Fig. 1 D-I). We validated the clock machinery dependency in vivo with Hep3B xenografts by showing that shRNA-mediated Bmal1/Clock knockdown strongly inhibited tumor growth ( Fig. 1 J and K). Therefore, the master circadian clock transcription factor heterodimer BMAL1::CLOCK plays a critical role in HCC cell proliferation, irrespective of the robustness of circadian oscillations or other genetic backgrounds, and is relevant to impaired tumor growth in vivo.

Targeting Bmal1 and Clock Induces Apoptosis and Cell Cycle
Arrest in HCC. To elucidate the molecular mechanisms underlying BMAL1::CLOCK-regulated HCC proliferation, we determined . Statistical significance was determined by Student's t test (**P < 0.01, ***P < 0.001). (E) Control and Bmal1 knockout mouse livers were harvested at 4-h intervals throughout 24 h. Transcript level of genes was analyzed by using RT-qPCR. Displayed are the means ± SD (n = 3 or 4) normalized to non-oscillating Rplp0 expression levels. P-values determined by two-tailed Student's t test were displayed (*P < 0.05, **P < 0.01, ***P < 0.001). cellular effects of Bmal1 and Clock knockdown. Flow-cytometric measurement of Annexin V and propidium iodide (PI) staining as well as the cleavage of CASPASE 3 revealed the activation of bonafide apoptosis upon Bmal1 or Clock knockdown (Fig. 2 A  and B and SI Appendix, Fig. S2). Bmal1/Clock-targeted HCC cells displayed a decreased G 1 fraction and a concomitant increase in the G 2 /M fraction relative to cells transduced with a non-targeting control siRNA (Fig. 2 C and D), indicating inhibited cell cycle progression. To determine if similar regulation occurs in normal tissues, we performed RT-qPCR characterization of the Bmal1 knockout mouse liver, which identified extensively disrupted transcription of cell cycle genes compared to the wild-type liver; specifically, Wee1 and Ccnb1 were down-regulated and p21 and Myc were up-regulated (Fig. 2E). Therefore, BMAL1::CLOCK may control transcription of the cell cycle regulators (20).

The Circadian Clock Contributes to Control of Cell Cycle Progression by Transcriptional Regulation of Ccnb1, p21,
and Myc. We were accordingly prompted to investigate whether BMAL1::CLOCK promotes the growth of HCC cells by modulating cell cycle progression and division. We first inspected the mechanism underpinning clock-regulated cell cycle progression. As cell cycle progression requires wellorchestrated transcriptional control of the cell cycle regulators (21), we characterized the impact of the altered transcription of cell cycle genes in response to Bmal1 depletion (Fig. 2E). siRNAmediated Wee1 downregulation induced G 1 phase accumulation (Fig. 3 A and B), which is compatible with its functional roles in activating the G 2 /M checkpoint (22). In contrast, the knockdown of Ccnb1, a G 2 /M-specific cyclin, reduced the fraction of cells in G 1 phase and increased the proportion of cells in the G 2 /M phase (Fig. 3 A and B). Overexpression of p21, a cyclin-dependent kinase inhibitor that promotes cell cycle arrest (23), increased the fraction of cells arrested in G 2 /M phase (Fig. 3 C and D). Consistent with previous studies (24), induction of Myc expression increased cell population arrested in the G 2 /M phase (Fig. 3 C   and D). Therefore, the G 2 /M phase cell cycle arrest resulting from Bmal1/Clock knockdown (Fig. 2 C and D) is likely associated with transcriptional downregulation of Ccnb1 and upregulation of p21 and Myc.  (Fig. 4  A-F). Also, upregulation of the proto-oncogene Myc is unlikely to lead to HCC proliferation inhibition. Therefore, the G 2 /M phase arrest does not necessarily result in reduced cell proliferation per se, in concordance with previous reports that depending on molecular context, either inhibition or activation of the G 2 /M checkpoint can induce cell death (25). Bmal1 knockout increased the basal level of p21 and reversed its peak expression from ZT0 to ZT12 (Fig. 2E). Gréchez-Cassiau and co-workers reported that the cyclic expression of p21 was controlled by the RORE-binding clock transcription factors REV-ERBs and RORs and up-regulated p21 was responsible for the decreased hepatocyte proliferation rate in the Bmal1 knockout mouse liver (26). We found that concurrent knockdown of p21 partially rescued HCC growth inhibition induced by Bmal1/Clock knockdown (Fig. 4 G and H). p21 overexpression did not substantially change the activity of apoptosis in Hep3B cells (SI Appendix, Fig. S3), in line with previous reports (27). Collectively, our findings indicate that BMAL1::CLOCK negatively regulates the RORE-containing cell cycle regulator p21 to maintain proliferation rate of HCC cells. Bmal1/Clock knockdown, we found that down-regulating Wee1 attenuated proliferation and caused apoptosis in HCC cells (Fig. 5  A-H). As depleting Bmal1 significantly reduced Wee1 levels in both mouse liver and Hep3B cells ( Fig. 2E and SI Appendix, Fig. S4), we asked whether Wee1 downregulation contributed to cell lethality resulting from BMAL1::CLOCK inhibition. Overexpression of Wee1 did not change the expression levels of Bmal1 and Clock (Fig. 5I) but could partially rescue cell growth inhibition caused by BMAL1::CLOCK downregulation (Fig. 5J). Wee1 transcription showed robust circadian oscillation in the mouse liver, peaking at the end of the day, in phase with the classical BMAL1::CLOCK target gene Dbp (Fig. 2E). BMAL1 chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) indicated that BMAL1::CLOCK binds Wee1 genes in both mouse liver and Hep3B cells (Fig. 5K). Therefore, Wee1 inhibits apoptosis and contributes to BMAL1::CLOCK-controlled HCC cell growth.

Discussion
Previous studies strongly support the crosstalk between HCC and the circadian clock. Modeling chronic jet lag to disrupt the circadian rhythms accelerated DEN-induced murine liver carcinogenesis (18) and even increased the incidence of spontaneous HCC associated with NAFLD (19). However, as chronic jet lag induced circadian desynchrony of most key clock genes, it is challenging to generalize from the results to the role of any single clock component in the observed HCC predisposition. Conversely, circadian transcript analysis of patient specimens revealed that a large body of genes cycling in noncancerous tissues, including specific core clock genes, lost oscillations in HCC tumors (29)(30)(31)(32)(33)(34). Here, our results provide molecular insights into this HCC-clock crosstalk by unveiling that the core clock transcription factor BMAL1::CLOCK is hijacked by cancer cells to fuel rapid cell proliferation and inhibit apoptosis in HCC, independent of the genetic background or core clock oscillations.
Mechanistically, inhibiting Bmal1 or Clock triggered systemic dysregulation of the cell cycle in HCC cells featured by activation of cell cycle arrest and apoptosis. Relative to Bmal1, we found knocking down Clock induced more apoptosis and arrested cell cycle at both the S and G 2 /M phases. It will be of interest for future studies to investigate whether the difference is because of potentially differentiated biological functions of the Bmal1 and Clock genes or solely due to higher knockdown efficiency of the Clock siRNA ( Fig. 1 G-I). Further, we provide genetic evidence supporting a scenario in which transcriptional alterations of the key cell cycle regulators Wee1 and p21 cooperatively contribute to cell growth inhibition in cells where BMAL1::CLOCK is down-regulated. BMAL1::CLOCK downregulation halted the cell cycle progression by up-regulating p21 and meanwhile activated apoptosis by down-regulating Wee1. As BMAL1 cooperates with the hepatocyte nuclear factor HNF4A in driving the liver-specific circadian Displayed are the means ± SD (n = 3 cell culture wells) normalized to Rplp0 expression levels. Statistical significance was determined by a two-tailed Student's t test (***P < 0.001). (G) Relative cell numbers of Hep3B transfected with scramble, Bmal1, Clock, or p21 siRNA. Data are represented as mean ± SD (n = 4). Statistical significance was determined by two-way ANOVA with Tukey multiple comparison test (***P < 0.001). (H) Transcript level of genes in Hep3B transfected with scramble, Bmal1, Clock, or p21 siRNA was determined by RT-qPCR. Displayed are the means ± SD (n = 3 cell culture wells) normalized to Rplp0 expression levels. Statistical significance was determined by a two-tailed Student's t test (**P < 0.01, ***P < 0.001; ns, not significant). regulation of tumorigenesis (15,16,35,36), BMAL1::CLOCK is a versatile player in HCC oncogenesis targeting multiple pathways. This study provides proof-of-principle for future development of novel liver cancer therapies via modulation of the circadian clock. CRYs, PERs, and REV-ERBs agonists inhibit BMAL1::CLOCK through circadian clock negative feedback loops (7). Therapeutics targeting the molecular clock has strong potential to be explored either as monotherapy or in combination with other therapies, with Wee1 and p21 potentially serving as molecular markers for evaluation of anticancer efficacy. p21, a tumor suppressor that arrests cell growth by inhibiting cell cycle progression, is expressed at lower levels in HCC tissues than that in the corresponding normal liver (37)(38)(39). p21 ablation induces continuous hepatocyte proliferation and enhances hepatocarcinogenesis in mice (40,41). Thus, controlling compensatory proliferation by high levels of p21 is likely critical to preventing HCC development. Pharmacological approaches developed for anticancer therapy targeting histone deacetylase (HDAC), PI3K-Akt, or MDM2-p53 interaction selectively induce p21 expression, thereby leading to decreased cell viability (42). Our findings shed new light on p21 regulation, now identifying the gene as a target of the circadian clock transcriptional network underlying the oncogenic function of BMAL1::CLOCK. Therefore, targeting p21 for activation of cell cycle arrest presents an opportunity to potentiate HCC therapy using clock drugs as an adjuvant. Normal cells repair damaged DNA during the G 1 /S cell cycle arrest, whereas cancer cells often have a deficient G 1 /S checkpoint and depend on a functional G 2 /M checkpoint for DNA repair and survival. The WEE1 kinase that plays a crucial role in the G 2 /M checkpoint arrest displays universally high expression in cancer. WEE1 inhibitors, including AZD1775 (MK1775), have been developed to compromise the G 2 /M checkpoint in combination with DNA damaging agents (22). In HCC tumors, WEE1 levels and kinase activity were found significantly elevated relative to surrounding cirrhotic tissues (43). Concordant with a previous study showing that BMAL1::CLOCK directly activates Wee1 transcription in an vitro system (44), we found that BMAL1 bound the Wee1 gene promoter (Fig. 5K) and Bmal1 depletion significantly reduced the Wee1 mRNA levels in liver cells (Fig. 2E  and SI Appendix, Fig. S4). Down-regulating Wee1 in HCC cells substantially attenuated HCC cell viability and induced apoptosis, which played an important role in the cytotoxicity effect of Bmal1/Clock inhibition (Fig. 5 A-J). Collectively, our observations support the concept that anti-apoptosis activity of the cancer-specific WEE1 contributes to BMAL1::CLOCK stimulated HCC cell proliferation. Small molecules targeting the clock are supposed to mimic WEE1 inhibitors but may provide increased specificity over the kinase modifiers for increasing DNA damage sensitivity of HCC patients.

Materials and Methods
Animal Experiments. All animal care and experiments were performed under the institutional protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Southern California. For mouse liver RT-qPCR analysis, mice were housed in a 12-h light/12-h dark (LD) cycle with free access to food and water and age of 10 to 12 wk were used. For the xenograft transplantation model, NU/J nude mice (The Jackson Library #002019) were subcutaneously transplanted in the right flank with 3 × 10 6 Hep3B cells transduced with a non-targeting control shRNA or shRNA targeting either Bmal1 or Clock. Tumor growth was monitored for 4 wk by measuring tumor diameter and weight at the endpoint.